Photosynthetic Antenna Size in Higher Plants Is Controlledby the Plastoquinone Redox State at the Post-transcriptionalRather than Transcriptional Level*□S

Received for publication, June 21, 2007, and in revised form, August 2, 2007 Published, JBC Papers in Press, August 5, 2007, DOI 10.1074/jbc.M705132200

Sara Frigerio‡§, Chiara Campoli¶, Simone Zorzan§, Luca Isaia Fantoni�, Cristina Crosatti¶, Friedel Drepper**,Wolfgang Haehnel**, Luigi Cattivelli¶, Tomas Morosinotto‡‡, and Roberto Bassi‡§1

From the ‡LGBP, UMR 6191 CEA-CNRS-Universite de la Mediterranee, Marseille 13288, France, the §DipartimentoScientifico e Tecnologico, Universita di Verona, Verona 37134, Italy, the ¶CRA-Centro per le Ricerche Genomiche,Fiorenzuola d’Arda 29017, Italy, the �Dipartimento di Scienze Biomediche, Universita di Modena e Reggio Emilia,Modena 41100, Italy, the **Institut fur Biologie II/Biochemie, Albert-Ludwigs-Universitat Freiburg,Freiburg D-79104, Germany, and the ‡‡Dipartimento di Biologia, Universita di Padova,Via Ugo Bassi 58 B, Padova 35131, Italy

We analyze the effect of the plastoquinone redox state on theregulationof the light-harvesting antenna size at transcriptionaland post-transcriptional levels. This was approached by study-ing transcription and accumulation of light-harvesting com-plexes in wild type versus the barley mutant viridis zb63, whichis depleted in photosystem I and where plastoquinone is consti-tutively reduced.We show that themRNA level of genes encod-ing antenna proteins is almost unaffected in the mutant; thisstability of messenger level is not a peculiarity of antenna-en-coding genes, but it extends to all photosynthesis-related genes.In contrast, analysis of protein accumulation by two-dimen-sional PAGE shows that themutant undergoes strong reductionof its antenna size, with individual gene products having differ-ent levels of accumulation.We conclude that the plastoquinoneredox state plays an important role in the long term regulationof chloroplast protein expression. However, its modulation isactive at the post-transcriptional rather than transcriptionallevel.

Sunlight is the only energy source for plants: light is absorbedby chlorophylls and carotenoids bound to the pigment-proteincomplexes composing photosystems I and II (PSI and -II),2 andit is converted into chemical energy. Both photosystems arecomposed by two distinct moieties: (i) a core complex, respon-sible for charge separation and for the first steps of the electron

transport, and (ii) an antenna system that increases light-har-vesting capacity. In photosynthetic eukaryotes, the antenna sys-tem is composed by the members of a multigenic family calledLhc (light-harvesting complexes) (1). To avoid the over-accu-mulation of excitation energy in photosystems, light-absorp-tion capacity needs to be related to the electron transport rate.To this purpose, the number of antenna proteins associated tophotosystems is regulated according to the environmental con-ditions (2). More recently it was shown that the regulation ofantenna size is restricted to PSII, whereas PSI-LHCI stoichiom-etry remains constant (3). Besides antenna size regulation,other mechanisms, like the state transition, the adjustment ofPSI/PSII ratio, and regulation of carotenoid biosynthesis, arealso involved in the plant’s long term acclimation to differentconditions (4). The limiting step of electron transport chainfrom PSII to PSI is the oxidation of plastoquinol (PQ) by cyto-chrome b6f (5); the PQ pool redox state depends on PSII exci-tation pressure, on the donor side, on the PSI capacity as accep-tor, and on the rate of PQ reduction by cyclic electron transport(6). Thus, the ratio between reduced and oxidized plastoqui-none is considered a good indicator of the balance betweenlight absorption and electron transport rate: PQ over-reduc-tion, in fact, suggests that electron transport is unable to use allthe energy absorbed by PSII, where accumulation of excitedstates easily leads to the formation of harmful reactive oxygenspecies (ROS) (7). Thus, it is not surprising that the PQ poolredox state has been identified as a key signal modulating theexpression of photosynthesis-related genes in response to lightconditions (8). Studies on algae like Dunaliella tertiolecta andChlamydomonas reinhardtii (9) showed that PQ redox stateinfluences dramatically the expression level of photosyntheticgenes. In particular, the largest effects were observed in thegenes encoding members of the lhc family, whose expression isstrongly repressed upon exposure to strong light. Besides plas-toquinone, also thioredoxin, glutathione, and ROS were pro-posed as signals for regulation of gene expression (for a reviewsee Ref. 10). The picture is evenmore complicated in the case ofmulticellular organisms like vascular plants, where the redoxsignal represents, in each cell, the state of 80–100 chloroplastsrather than a single organelle (10, 11).

* This work was supported by the Provincia Autonoma di Trento, progettoSAMBAx2 and Progetto Fondo Investimenti Ricerca di Base PARALLELOMICS(RBIP06CTBR). The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental files and tables.

1 To whom correspondence should be addressed: Dipartimento Scientifico eTecnologico, Universita di Verona, Strada Le Grazie 15, 37134, Verona, Italy.Tel.: 39-045-802-7916; Fax: 39-045-802-7929; E-mail: bassi@sci.univr.it.

2 The abbreviations used are: PSI, -II, photosystems I and II; �(�)-DM, n-dode-cyl-�(�)-D-maltoside; Lhca (b), light-harvesting complex of PSI (II); LHCI,antenna complex of PSI; PQ, plastoquinone; ROS, reactive oxygen species;WT, wild type; Rubisco, ribulose-bisphosphate carboxylase/oxygenase; RT,reverse transcription; MS, mass spectrometry; HPLC, high-performance liq-uid chromatography; SAM, significance analysis on microarray; E, einstein.

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Despite these studies, the role of gene expression regulation inlong termacclimationof thephotosynthetic apparatus todifferentlight conditions is still unclear. In fact, while the regulation of thepolypeptide level was reported by several laboratories (2, 12), datafor mRNA level are still incomplete. This lack of information isprobably due to the fact that treatments with electron transportinhibitors, likeDCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea)and DBMIB (2,5-3-methyl-6-isopropyl-p-benzoquinone),widely used to simulate different PQ redox conditions, cannot beprolonged indefinitely. In addition, it was recently shown that therepression of lhc gene transcription induced by strong illumina-tion is reversible after �24 h, even if the light treatment is main-tained, thus suggesting that regulation mechanisms could be dif-ferent on the longer termwith respect to short term stress (13).In this work we analyzed the regulation of antenna system in

barley mutant viridis zb63, which has a chronically over-re-duced PQpool even if grown at very low light intensities (14), toobtain new insights on the role of plastoquinone redox stateboth on gene and protein long term regulation. The use of thismutant allows the study of a long termPQover-reductionwith-out the need of inhibitor treatments. Viridis zb63 was recentlyshown to have a PSII antenna system reduced to its minimumlevel: a dimeric PSII reaction center core surrounded by onecopy of each monomeric Lhcb4, Lhcb5, and the trimeric light-harvesting complex II (14).We found that, despite these strongeffects on Lhc protein accumulation, transcription of corre-sponding genes was substantially unaltered in the mutant, ascompared with WT, whose PQ is oxidized in low light (14).Stability of transcription level was not restricted to lhc genes,but it was observed for most photosynthesis-related genes. ThePQpool redox state, thus, plays a role in long term regulation ofthe expression of antenna proteins mainly at the post-tran-scriptional level, whereas its effect on gene transcription is rel-evant only a few hours after alteration in light conditions.

EXPERIMENTAL PROCEDURES

Genetic Material and Growth Conditions—A spring barley(Hordeum vulgare cv Bonus) and a mutant lacking PSI, viridiszb63, obtained by chemical mutagenesis on the cv Bonusgenetic background, were used. Plants were grown for 9 days at23 °C with 8-h photoperiod and 100 �mol m�2 s�1 of light. ForRNA extraction leaves, in the middle of the light period, wereharvested and immediately frozen into liquid nitrogen. Thisexperiment was conducted three times to obtain three inde-pendent biological replicates.RNA Isolation and Array Hybridization—Total RNA was

prepared using TRIzol reagent according to the method pub-lished at theArabidopsis functional genomics consortiumweb-site and further cleaned using an RNeasy Minikit (Qiagen,Valencia, CA) following the manufacturer’s instructions. Dis-posable RNA chips (Agilent RNA 6000Nano LabChip kit) wereused to determine the concentration and purity/integrity ofRNAsamples usingAgilent 2100 bioanalyzer (AgilentTechnol-ogies, Palo Alto, CA).Double-stranded cDNA was synthesized starting from 5 �g

of total RNA using the GeneChip� Expression 3�-amplificationreagents-One Cycle cDNA Synthesis Kit (Affymetrix) accord-ing to the standard protocol. This double-stranded cDNA was

used as template to generate biotin-labeled cRNA from an invitro transcription reaction (IVT), using the GeneChip�Expression 3�-amplification reagents-For IVT Labeling Kit(Affymetrix) following manufacturer instructions. The result-ing biotin-labeled cRNA was fragmented into strands of35–200 bases in length following Affymetrix protocols, and 15�g of fragmented target cRNAwas hybridized on anAffymetrixBarley Genome Array according to the standard protocol (16 hat 45 °C with rotation in the Affymetrix GeneChip Hybridiza-tion Oven 640). The arrays were washed and stained on anAffymetrix Fluidics Station 450 according to the standard pro-tocol, then scanned on a Affymetrix GeneChip Scanner 3000.Data Analysis—Scanned images were analyzed using the Gene

ChipOperatingSoftware1.4 (Affymetrix).Expressionanalysiswasdone using default values. Quality control values, present calls,background, noise, scaling factor, spike controls, and the 3�/5�ratios of glyceraldehyde-3-phosphate dehydrogenase and tubulinshowed low variation between samples (15). Raw data files (CELfiles)werebackground-adjustedandnormalized, andgeneexpres-sion values were calculated using Robust Multichip Analysis(RMA)algorithm implemented in the statistical packageR2.3.1 (Rfoundation) with the dedicated affy library (R foundation). Nor-malized datawere imported into theGenespringGX7.3.1 (SiliconGenetics) software for analysis. Each gene was normalized to themedian of themeasurements.Weused detection calls (present) asan initial filtering step: a present-absent filter was applied toremove probe sets with less than two present cells. A second filterwas applied to eliminate probe sets with normalized signal valuebetween 0.66 and 1.5, to evaluate significant the -fold change.In the comparison (wild type versus viridis zb63) the baseline

was set as wild type; genes with at least a 2-fold change value inthe comparison were further considered. Lists of differentiallyexpressed genes were analyzed for statistical significantchanges by a Welch t test (analysis of variance) with the Ben-jamini and Hochberg false discovery rate correction; the falsediscovery rate-adjusted p value cutoff was set to 0.05. For thefunctional probe sets annotation BLAST search results wereexported from HarvEST 1.50.RT-PCR—RT-PCRwas performed both on nuclear and chlo-

roplast genes. For each reaction, first strand synthesis was doneon 0.5�g of total RNA (treatedwithDNase I) using 200 units ofMoloney murine leukemia virus reverse transcriptase (Sigma)and a poly(T) primer, for nuclear genes, or a gene-specificprimer, for chloroplast sequences. This synthesis was followedby RNase H treatment.Transcripts were then amplified using the following prim-

ers: nuclear genes: lhca3 (Contig1815_at), 5�-GCAGTACT-TCCTGGGTCTCGAGAAG-3� and 5�-GCAATGCACTA-ATTTTCATAGCACGATA-3�; lhcb1 (Contig422_at), 5�-AGTTGCTTCGAGCAGCCCGTGGTA-3� and 5�-AGTG-GGCCACCGGCAATGCGGTA-3�; lhcb6 (Contig1523_at),5�-AAGCGATGCCGAGTTCATCAACCCGT-3� and 5�-TAC-CCTTGCTCGCCGGTGAAGTTG-3�; psaH (Contig2250_at),5�-CAGCTCGACCGCAATGGCGTCGCT-3� and 5�-CGCG-GCGAAGGTGTTGAAGAACTTGC-3�; �-carotene desatu-rase (Contig HW04B18_u_s_at), 5�-CTCAAGGTCGCCATC-ATAGGTGCC-3� and 5�-GATCAACAAGAGCTTGAACAA-CTGGGC-3�; glycerophosphoryl diesterase (Contig 8049_at),

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5�-TCGACTACGTCGAGTTCGACGTGC-3� and 5�-AGTT-TTCGCATGAGTTGCGCAGCATCG-3�; and copper-bind-ing protein (Contig 17974_at), 5�-CCTTTTCACTTCAGAG-CTTCACTGTTC-3� and 5�-AGCACCTCATAGGGCGTC-ACGTC-3�. The chloroplastic genes were: psaA (HV_CEa0013J19f_at), 5�-CTTTATCCTAGTTTTGCCGAAGG-AGCA-3� and 5�-GGTATGGATAGGGAGGCATAGAAT-ACA-3�; psaC (HV_CEb0010M11f_at), 5�-GTCTCTTGGC-TCTTTTCACGCTTTCT-3� and 5�-CATGCTACGGGTT-GTTTCAGGCCCT-3�; psbA, 5�-TCCCTATTCAGTGCT-ATGCATGGTTCC-3� and 5�-TCAACAGCAGCTAAGTC-TAGAGGGAAG-3�; and psbD, 5�-GATGGGAGTTGCCG-GAGTATTAGGC-3� and 5�-CATGAGGCTGATCCT-GAGCTGCCAT-3�.The transcripts were amplified from 50 ng of cDNA as tem-

plate and 2.5 units of TaqDNA polymerase (Sigma) using thefollowings cycles: 94 °C for 30 s, annealing for 30 s, 72 °C for40 s, followed by a final extension step at 72 °C for 1 min. Tohighlight the exponential phase, the amplification was stoppedafter 24, 26, and 28 cycles, and 5 �l for each gene was collected.RT-PCR reactions were performed with primers designed ongene coding with �-actin used as the standard. Amplificationproducts were separated on 2% agarose gel.Thylakoid Isolation and Quantification—From 9-day-old

plants, thylakoidswere isolated as in a previous study (16). Afterthe isolation, the membranes were further purified through acentrifugation on a sucrose step gradient: thylakoids wereresuspended in 1.9 M sucrose, 25 mM Hepes, pH 7.5, 10 mMEDTA, and protease inhibitors then stratified at the bottomof astep gradientmade in SW28 tubeswith 1.3M and 1.14M sucrosesolutions. After 1-h centrifugation at 27,000 rpm, the big greenband containing purified thylakoids was recovered from theinterface of the step gradient. Thylakoids were quantified boththrough chlorophyll absorption spectra and using a BCA assay(Sigma-Aldrich) for total protein content.ProteomicAnalysis—Proteins from thylakoidswere prepared

for isoelectric focusing as follows: first, proteins were de-phos-phorylated by treatingwith calf intestine phosphatase) for 1 h at37 °C. Then, after the precipitation with 14 volumes of tribu-tylphosphate:acetone:methanol (1:12:1, v/v), proteins wereresuspended using 7 M urea, 2 M thiourea, 40mMTris, 1% amido-sulfobetaine-14, 1% carrier ampholyte, pH 3–8, reduced with 5mM tributyl phosphine, and alkylated with 10 mM acrylamide.Protein solution obtained was then used for passive re-hy-

dration of the immobilized pH gradient strip (pH range 3–8,from Bio-Rad), then focused (3 h with linearly increasing volt-age till 5 KV, then 5 KV till VH reach 65,000). The seconddimension was performed with a gel system described before(17) with acrylamide gradient 12–18%. Gels were stained withSypro Ruby (Sigma-Aldrich), and images for quantitativeanalysis with PDQuest software where acquired with a Versa-Doc detection system (Bio-Rad). Spots where cut from gels forMS analysis: the proteins from punched out spots weredestained, alkylated with 55 mM iodoacetamide after reductionwith 10 mM dithiothreitol, and digested in gel with trypsin at37 °C overnight, and the peptides were extracted with 1% for-mic acid. For desalting, the solution was transferred to a nano-HPLC column and finally eluted from a 75-�m, 70-mmcolumn

with reversed-phase C18 material with a gradient of 0–80%acetonitrile in 0.1% trifluoroacetic acid. Themass spectrometerwas a LTQ-FT (Thermo-Finnigan) with a linear ion trap and anion cyclotron with a 7-Tesla magnet.Lhcb1 Cloning and Expression—Sequences for different

Lhcb1 where obtained from a cDNA library, amplified, andcloned in a pQE50His vector, using BamHI and HindIII asrestriction sites (18). Transformed Escherichia coli cells(SG13009) where grown for 3 h at 37 °C then induced for 6 hwith 1 mM isopropyl 1-thio-�-D-galactopyranoside; the recom-binant proteinswhere purified as inclusion bodies following theprotocol from Nagai and colleagues (19).Reconstitution Procedure—The pigment-protein complexes

refolding in vitrowere performed as described before (20) withthe following modifications: the 1.1-ml reconstitution mixturecontained 420 �g of apoprotein, 240 �g of chlorophylls, and 60�g of carotenoids. The chlorophyll a/b ratio of the pigmentmixture was 2.3. All pigments used were purified from spinachthylakoids.Analysis of Reconstituted Complexes—High performance liq-

uid chromatography analysis was performed as describedbefore (21). Chlorophyll to carotenoid ratio and chlorophyll a/bratio were measured independently by fitting the spectrum ofacetone extracts with the spectra of individual purified pig-ments (22). The absorption spectra at room temperature wererecorded using a SLM-Aminco DK2000 spectrophotometer, in10 mM HEPES, pH 7.5, 0.5 M sucrose, and 0.06% n-dodecyl-�-D-maltoside. The wavelength sampling step was 0.4 nm, scanrate 100 nm/min, and optical path length 1 cm.Fluorescence emission spectra were measured using a

Jasco FP-777 spectrofluorometer and were corrected forinstrumental response. For emission spectra, samples wereexcited at 440, 475, and 500 nm with spectral bandwidths of5 nm (excitation) and 3 nm (emission). For excitation spec-tra, emission was recorded at 685 nm, with bandwidths of 3and 5 nm for excitation and emission, respectively. Chloro-phyll concentration was �0.02 �g/ml in 10 mM HEPES, pH7.5, and 0.03% n-dodecyl-�-D-maltoside.Western Blot Titration—For protein quantity titration using

Western blot assay, thylakoids were loaded on minigels usingthe system described in a previous study (17), in four differentdilutions to achieve a good reproducibility and avoid saturationeffects. Different samples were quantified for their protein con-tent using a BCA assay. For viridis zb63, on gels for Lhca3,Lhca4, Lhcb1, Lhcb4, Lhcb5, Lhcb6, PsbB, and PsbC, assayswere loaded 11, 22, 33, and 44 �g of total proteins, whereas forLhcb3, PsaE, and PsaF the protein amount was reduced to 1.2,2.2, 4.4, and 8.8 �g of proteins. Similarly, for viridis 115 on gelsfor Lhcb4 and Lhcb6, assays were loaded 4, 8, 12, and 16 �g ofproteins, whereas for LHCII the amounts were 0.4, 0.8, 1.6, and3.2 �g of proteins. One microgram of protein corresponded to0.10 �g of Chls, 0.04, 0.06 per WT, Viridis Zb63 and 115,respectively. The results of the antibody reactionwere acquiredwith a scanner and analyzed by densitometry using Gel-ProAnalyser 3.1.Non-denaturing Deriphat-PAGE—Non-denaturing Deriphat-

PAGE was performed as in a previous study (23) with the fol-lowing modifications: the stacking gel had 3.5% (w/v) acrylam-

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ide (48:1.5% acrylamide/bisacrylamide). The resolving gel hadan acrylamide concentration gradient from 4.5 to 11.5% (w/v)stabilized by a glycerol gradient from 8% to 16% (w/v). 12 mMTris and 48mM glycine, pH 8.5, were also included in both gels.Thylakoids, at chlorophyll concentration of 1 mg/ml, were sol-ubilized with an equal volume of 1.6% of �-DM (Anatrace�,Maumee, OH). Solubilized thylakoids were vortexed for 1 min,left for 10 min in ice, and then centrifuged at 13,000 rpm for 15min for pelletting un-solubilizedmaterial. 30�g of chlorophyllswas loaded per each gel lane.Biochemical Determination of Lipid Peroxidation—Lipid

peroxidation was assessed indirectly by measuring malondial-dehyde using HPLC, as before (24), with the following modifi-cations. The samples (each one consists of 3 leaf discs of 5 mmin diameter) were exposed to 100 �E (control) and to 1300 �Elight (high light) or incubated in the dark, for 4 h. After incuba-tion, sampleswere frozen in liquid nitrogen, then ground in 150�l of 175 mM NaCl and 50 mM Tris-HCl, pH 8.0. A volume of0.5%w/v thiobarbituric acid in 20%w/v trichloroacetic acidwasadded, and samples were incubated at 95 °C for 25 min. After20-min centrifugation, the malondialdehyde(thiobarbituricacid)2 adductwas separated and quantified byHPLC, using 65%50 mM KH2PO4-KOH, pH 7.0, and 35% methanol as elutionbuffer.

RESULTS

Differences in Transcription Rate between WT and Viridiszb63 Plants Grown in Control Conditions—Viridis zb63 is abarleymutant depleted in PSI but retaining a normal PSII activ-ity (25, 26). The mutation is lethal, and it is maintained in aheterozygous state. Homozygous mutant plants grow up to 2weeks on seed reserves, and they are easily distinguishable fromWT because of their paler green color. The mutation causes a

block of linear electron transport,which was shown to cause thechronic over-reduction of the PQpool even in low light (20 �E) con-ditions (14). In this work we grewWT and mutant plants in dim lightconditions (100 �E) ensuring thecomplete reduction of the PQ poolin the mutant but not in the WT.Because viridis zb63 is depleted inPSI but still presents a residual 2%PSI activity (26), we verified thedependence of PQ redox state onlight intensity in WT versusmutantleaves. As shown in Fig. 1, left panel,even very low illumination causes asaturation of PSII reaction centersin the mutant. On the contrary, inthe WT saturation is achieved onlywith far stronger light intensities.This measure, thus, demonstratesthat in growing conditions usedhere PQ pool is completely reducedin the mutant but not in the WTplants.

The same light intensity was also chosen, because it did notcause significant oxidative stress in themutant: as shown in Fig.1, right panel, the amount of lipid peroxides generated at 100�eq with respect to dark conditions is similar inWT and viridiszb63, whereas larger accumulation of lipid peroxides is insteadobserved both in WT and mutant when leaves are exposed tohigh light (1300 �E), where the plastoquinone pool is reducedin WT plants as well.The redox state of PQ and other electron transporters was

proposed to play a key role in regulating the expression of pho-tosynthetic genes (8, 27). However, these studies were allrestricted to the effects of few hours of light treatment. In thiswork we used viridis zb63 as amodel to identify genes respond-ing to the PQ redox state on a longer timescale, by comparingthe mutant transcriptome to the wild-type one. After 9 days ofgrowth at 100 �E, total RNA was extracted from plants andretro-transcribed using the poly(A) tail. The correspondingbiotin-labeled cRNA was hybridized to the 22 k Barley 1 Gene-Chip (15) on three biological replicates for each genotype. A2-fold change cut-off was applied, followed by analysis for sta-tistically significant changes using Welch-t test and SAM (Sig-nificance Analysis on Microarray). Leaves used for samplingwere maintained under continuous illumination during har-vesting; this was an essential precaution in the case of themutant, because we observed that the PQ redox pool is com-pletely reduced even by very low illumination, but it is re-oxi-dized, possibly by alternative chloroplastic oxidases, after fewminutes of darkness (data not shown).In the described experimental conditions a total of 542 genes

of 22,840 (from here forward we will refer to probe sets asgenes) were differentially expressed between WT and mutantplants; in particular, 503 geneswere up-regulated and 39 down-regulated in viridis zb63 with respect to WT. Their classifica-

FIGURE 1. Determination of PQ redox state and lipid peroxidation in WT versus viridis Zb63 plants. Left,determination of PSII (PSII) efficiency (�PSII) in wild-type (WT, squares) and mutant (circles) leaves illuminatedwith using increasing light intensities. Right, the reaction between ROS and lipids drives the accumulation ofmalondialdehyde (MDA): the graph shows the malondialdehyde-(thiobarbituric acid)2 adduct formation in WTand viridis zb63 (zb63) leaf discs, after exposure to different light intensities.

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tion is reported in Fig. 2; the complete list of up- and down-regulated genes is reported in the supplemental material.Among them, 168 probe sets were annotated as an

“expressed protein” of unknown function or as new genes with-out correspondence in the databases. 76 of the remaining genes(73 of which up-regulated) were associated to RNA transcrip-tion and maturation. 42 genes (36 up-regulated) were insteadassociated to protein synthesis or protein degradation, likeubiquitin and proteases. In addition, 16 genes related to cellcycle were up-regulated, including senescence-associated fac-tors, apoptosis, and programmed cell death involved proteins.In themutant, 53 genes involved in signalingwere also induced,like kinases and phosphatases as well as 13 factors implicated inhormone biosynthesis or response. As expected, the mutationaltered metabolism as well: in particular, 31 enzymes involvedin sugar metabolism and translocation as well as 22 enzymes oflipid biosynthesis were up-regulated. Eighteen known stress-related genes were regulated and four are known to be involvedin oxidative stress response: two peroxidases, one ferritin (28)and a protein induced by radicals (29). This result is consistentwith previous data suggesting that mutant plants experience achronic light excess condition, as confirmed by the large accu-mulation of zeaxanthin (14, 30).As mentioned, it was shown that PQ is over-reduced in viri-

dis zb63 even in low light conditions (14). Because the PQ redoxstate was shown to have a regulatory role in the acclimation ofphotosynthetic apparatus to different light conditions (8), weexpected the mutant to have an altered expression of genesinvolved in the light phase of photosynthesis. On the contrary,among the differentially expressed genes only one could beidentified as photosynthesis-related, and it does not encode aprotein of the light-harvesting complex, but a �-carotenedesaturase, an enzyme involved in the first steps of carotenoidbiosynthesis. This observation is surprising if we consider that

the composition of the photosynthetic apparatus of viridis zb63is largely modified with respect toWT. To get new insights, weanalyzed in deeper detail the original data set for the expressionlevel of all genes encoding PSI, PSII, and cytochrome b6f sub-units (31, 32); the complete list of these genes considered asinvolved in the light phase of photosynthesis identified in thearray, together with their expression level, is reported in thesupplemental material.With the mentioned exception of �-carotene desaturase,

which is expressed only 2.2 timesmore in themutant, the other231 genes considered show only small variation, below 2-fold,in expression between the two genotypes. In the few caseswhere average values are over the 2-fold limit, standard devia-tion is also higher and the statistical analysis classified thesedifferences as non-significant with a p value of 0.05. This con-clusion is also valid for chloroplast ATPase subunits: in fact,only one gene showed an altered expression in the mutant, andits sequence clearlymisses any plastid localization peptide, sup-porting the conclusion that ATPase-encoding sequences arealso not regulated in the experimental conditions.Because this result was really unexpected, considering pres-

ent literature in the field, we looked more closely to these 231photosynthesis-related genes. We found that they can be dis-tinguished in two classes: the majority, a total of 181, show alogarithmic -fold change of��0.5, thus they can be consideredas unchanged. The remaining 46 genes, instead, are slightlyregulated with a -fold change between 0.5 and 1. Interestingly,in all 46 cases the -fold change was negative, thus indicating amoderate repression. The fact that this regulation does notovercome the cut-off threshold could depend on experimentaldesign, which does not allow discriminating confidently suchsmall changes. However, the fact that all 46 genes are consis-tently repressed excludes the possibility that all differences aredue to stochastic effects. These data thus support the view that,although the largest part of photosynthetic genes is not regu-lated, a sub-class is insteadmoderately repressed in themutant.Among the latter we found 14 sequences encoding for Lhc pro-teins and in particular 12 encoding Lhcb1. These representone-half of the total probe sets for this subunit and indicate thepresence of a significant effect of the mutation, even if smallerthan expected, on the regulation of Lhcb1 genes transcription.The remaining two regulated probe sets correspond to Lhcb4and Lhca1: because for these polypeptides we found, respec-tively, another three and four unchanged probe sets, we cannotaffirm confidently that they are regulated, and, even if such aregulation is present, it is surely smaller than in the case ofLhcb1.To verify the quality of the array experiments and confirm

the surprising absence of a significant variation in photosynthe-sis-related genes expression,we analyzed the expression level ofsome sequences by semi-quantitative RT-PCR. For this assaywe chose four nuclear-encoded genes (lhca3, lhcb1, lhcb6, andpsaH) among the different polypeptides composing both thecore and the antenna system of PSI and PSII, the only photo-synthesis-related sequence showing variation in microarrayanalysis (encoding �-carotene desaturase), one random genestrongly up-regulated and one down-regulated in the mutant(encoding, respectively, a glycerophosphoryl diesterase and a

FIGURE 2. Classification of up- and down-regulated genes in the viridiszb63 mutant with respect to WT. Pie graphs show a total of 503 up-regu-lated and 39 down-regulated genes, classified on the basis of their putativefunctional role.

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copper-binding protein). The RT-PCR analysis (Fig. 3A) con-firmed the absence of big differences in the photosynthesis-related genes, but in the case of �-carotene desaturase, which isunder-regulated in the WT, glycerophosphoryl diesterase andthe copper-binding protein also confirmed their large alter-ation of expression. In the case of Lhcb1, we observed a verysmall decrease of expression in themutant, thus in the expecteddirection. Considering all the probe sets representing Lhcb1,the medium -fold change between WT and viridis zb63 wasonly�0.43. Furthermore, in this case PCR analysis was compli-cated by the fact that several very similar Lhcb1 isoforms werepresent in the barley genome and they were probably notequally amplified during the reaction, possibly smoothing smallvariations in transcript levels. Even considering the limitationsin the case of Lhcb1, however, PCR results are in good agree-ment with array experiments.Several polypeptides composing the photosynthetic appara-

tus, especially the photosystems reaction centers, are encodedby the chloroplast genome, but they are excluded from arrayanalysis because the total mRNA preparation exploits thepoly(A) tail for retro-transcription. Therefore, we found it use-

ful to verify whether our results could be explained by a differ-ent regulation of gene expression in the chloroplast and thenucleus. To verify this point, we evaluated the expression offour chloroplast-encoded sequences (two for each photosys-tem, psaA, psaC, psbA, and psbD) by RT-PCR as explainedabove, with the only difference of the individual retro-tran-scription for each gene. Also �-actin was individually retro-transcribed as the internal standard. No significant variation ofgene expression regulation was observed between WT and themutant (Fig. 3B). Thus, even considering the possible stochasticeffects in PCR reactions, these results strongly support thehypothesis of a similar effect in the regulation of both chloro-plast and nuclear photosynthetic genes.It was previously shown that the viridis zb63 mutant is

depleted in PSI complex, but this mutation is not due to theabsence of some of PSI polypeptides but to a still unknownfactor for PSI assembly (31). In addition to what was observedso far, it is also interesting to point out that, despite the absenceof fully assembled PSI complex and themissed accumulation ofany core polypeptides, all genes encoding PSI subunits are sub-stantially expressed at the same level with respect to wild-typeplants.Proteomic Analysis of Thylakoid Membranes—The results

from transcriptome analysis appeared to be in striking contrastwith a large number of previous reports showing the regulationof photosynthetic polypeptides during acclimation to differentlight conditions (2, 12). In the very same mutant, viridis zb63,the antenna content was recently shown to be strongly reducedeven in the relatively low light conditions used in this work.Furthermore, it was shown that antenna size in the mutant isreduced to theminimum possible level, with Lhcb6 being com-pletely absent and Lhcb1–3 strongly reduced. To further detailthe difference between gene expression and protein accumula-tion level, we performed a proteomic analysis specificallyfocused on Lhc polypeptides. Polypeptides in thylakoid mem-branes from wild-type and mutant plants were separated bytwo-dimensional isoelectric focusing-SDS-PAGE. The com-parison of different maps showed the presence of many spotsless abundant (at least 2-fold) in mutant plants with respect towild type, in the gel region corresponding to antenna proteins(Fig. 4). Western blot analysis with a mixture of antibodiesagainst Lhc proteins confirmed that no Lhc proteins migratedout of the gel area analyzed in detail (data not shown).All the spots visible in this region were cut and analyzed by

LTQ-FT (hybrid linear ion trap-Fourier transform-ion cyclo-tron resonance) mass spectrometry for identification. Digestedpeptides were first separated by HPLC and then their massdetermined at high accuracy with by the Fourier transform-ioncyclotron resonance. All peptides were fragmented and ana-lyzed byMS-MS scan at low resolution with the linear ion trap,allowing determination of peptide sequence. The high massaccuracy (�0.6 ppm) allows the discrimination of the individ-ual superimposed traces of the peptides, which was critical todistinguish between very similar polypeptides. One example ofsuch identification in the case of two peptides differing in onlyone amino acid (Asp in place ofGlu) is shown in Fig. 5. Accuratemass determination together with the detection of an almostcomplete series of the N-terminal B-fragments and the C-ter-

FIGURE 3. Expression analysis by semi-quantitative RT-PCR of selectedgenes in WT and viridis zb63 (zb63). for each gene the amplification reac-tion was stopped after 24, 26, and 28 cycles. A, selected nuclear genes;B, chloroplast-encoded sequences. �-Actin was used as control for the totalRNA amount.

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minal Y-fragments allowed unambiguous identification of thepeptide sequence. Because identified peptides covered a largepart of polypeptide sequences, it was thus possible to distin-guish Lhc isoforms from one another, even if they differed inonly a few amino acids.The complete list of identified proteins overexpressed

both inWT and in viridis zb63 is reported in the supplemen-tal material. Among the few proteins overexpressed in themutant, we could identify different oxygen-evolving complexsubunits, one ascorbate peroxidase, the ATPase �-chain, a plas-toquinone, and an NADH oxidoreductase. On the contrary,Lhca and Lhcb polypeptides were largely represented amongthe proteins underexpressed in themutant, confirming that theantenna size is extensively regulated in this mutant.To validate proteomic data, an immunotitration for different

subunits was performed both for PSII and PSI antenna and coreproteins. Thylakoids purified fromWTandmutant plants wereanalyzed by SDS-PAGE and immunoblotting using specificantibodies. To avoid saturation effects, four different dilutionsof each samplewere loaded in the same gel (3). BecauseWTandmutant thylakoids have different chlorophyll to protein ratio,

samples were normalized to theirtotal protein content. In the case ofPSII proteins, the assay for Lhcb1confirmed a 2-fold down-regulationin viridis zb63 with respect to WT.On the contrary, Western blots forLhcb3 showed a stronger reduction:only traces of this subunit weredetectable in the mutant mem-branes and only upon prolongeddetection, when other signals werealready saturated. Among minorantennas, Lhcb6 polypeptide wasconfirmed to be completely absentin the mutant, whereas Lhcb4 andLhcb5 did not show significantchanges between the two genotypes.Similarly, core proteins PsbB andPsbC were substantially unchangedin WT versus mutant. We were notable to detect PSI core subunitsPsaE and PsaF, confirming theirdepletion in viridis zb63, in agree-ment with a previous report (14).On the contrary, the assay for Lhca3and Lhca4 showed that these PSIantenna proteins were retained inmutant thylakoids despite thedepletion of the PSI core, althoughit was at least three times lower thanin wild type (Fig. 6).To verify if the retaining of

antenna polypeptides despite thecore absence is a unique PSI feature,we also verified the presence ofLhcb polypeptides in a different bar-ley mutant, viridis 115, which lacks

PSII core complex. Western blot assays showed that bothLHCII and theminor antennas Lhcb6 and Lhcb4 proteins weresignificantly accumulated, although to a reduced level withrespect to WT. This result confirms that all antenna proteinsare assembled even in the absence of the core complex both forPSII and PSI (Fig. 7).After validation of MS results, we verified the case of Lhcb1

polypeptides in viridis zb63 in deeper detail, because in barleythere are at least fourteen different isoforms (18). These pro-teins are very similar to each other on the basis of sequenceinformation, and it was not possible to completely distinguisheach individual isoform based on peptides obtained by MS.However, we were able to discriminate five different classes,each containing one to three different polypeptides and indi-cated as Lhcb1a, Lhcb1b, Lhcb1c, Lhcb1d, and Lhcb1e. As inthe case of array analysis, MS results showed that different iso-forms have different expression profiles: many Lhcb1 areunder-accumulated in the mutant, whereas a few others areretained at the same level than in wild type. In particular, wefound that polypeptides belonging to classes from Lhcb1b toLhcb1e were underexpressed in viridis zb63, while those of

FIGURE 4. Two-dimensional isoelectric focusing-SDS-PAGE analysis of WT and viridis zb63 (zb63) thyla-koids. The gel area containing Lhc proteins is shown. A, Sypro Ruby-stained gel of WT and viridis zb63. B, thesame gels with indication of spots overexpressed either in WT or in viridis zb63 (marked with an arrow). The listof polypeptides identified in each spot by mass spectrometry is reported in the supplemental material. On thegel from mutant thylakoids, the spot corresponding to the only unchanged spot corresponding to a Lhcb1isoform is also highlighted.

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class Lhcb1a were retained. It would be interesting to verify ifmembers of the Lhcb1a class present also mRNA expressionlevels different from the other isoforms. Unfortunately, iso-forms are so similar to each other that it was not possible toestablish a direct correspondence between Lhcb1 classes iden-tified at the polypeptide level and array data sets, and thus weare obliged to leave the question open.Nevertheless, the behavior of Lhcb1 is particularly interest-

ing because it suggests that different isoforms are independ-

ently regulated, possibly because they fulfill specific physiolog-ical functions; for this reason they can be either retained ordegraded in an over-reduced PQ genotype. Only two proteinsbelonged to the “retained” class: Lhcb1a.01 and Lhcb1a.02.These two sequences differ from all the others by three aminoacid residues only, apart from the signal peptide (sequencealignment is reported in the supplemental material): Lhcb1a.01carries Asn rather than Lys in position 91, Lhcb1a.02 carries Serversus Ala in position 210, and both proteins carry Glu instead

FIGURE 5. Example of spot identification by mass spectrometry. HPLC-MSdata for tryptic digest of spot wt21. HPLC chromatogram for Base peak (A) andthe following selected ions (B). Lines 1 (m/z 793.4095) and 2 (m/z 800.4172)identify two isoforms with Asp-202 and Glu-202 of the doubly charged Lhcb1peptide (197–211) PLGLA(D/E)DPEAFAELK with exact masses of Mr 1584.8044and Mr 1598.8198, respectively, as detected by selected ion monitoring-MS (C, leftand right spectra, respectively). Peptides labeled 1 and 2 are fragments of thelarger peptides 3, 4, and 5 with a missed trypsin cleavage site. Exact masses arem/z 1176.2592 (3), m/z 1180.9319 (4), isobaric sequences, with Ile-177 to Asp-202and Val-177 to Glu-202, both identified by MS/MS spectra (not shown), and m/z1185.6039 (5). All peptides were fragmented and the MS-MS scan at low resolu-tion in the linear ion trap. Sequence identification of C-terminal Y- and N-terminalB-ions for peptides 1 and 2 are shown in D and E, respectively.

FIGURE 6. Western blot titration on viridis zb63. Different polypeptideswere quantified in WT and viridis zb63 thylakoids by detection with specificantibodies. For each assay only one dilution is shown in A for clarity, but thequantification reported in B is based on four dilutions for each assay. All val-ues reported are normalized to protein content in WT.

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ofAsp in position 168.According to sequence analysis (1), thesesubstituted residues should not be involved either in chloro-phyll, in carotenoid binding, nor in trimer formation.Lhcb1 Reconstitution—To verify the hypothesis of a different

functional role for Lhcb1 isoforms, their cDNAs have beenamplified from a phage library and have been cloned in pQE50(18). One representative polypeptide was chosen for each of thefive classes, but in the case of the potentially most interestingclass, Lhcb1a, both isoforms were analyzed. All polypeptideswere expressed in E. coli, purified as inclusion bodies, andrefolded in vitro using a well established procedure (20). Pig-ment-protein complexes thus obtained have been analyzedspectroscopically and biochemically. In Fig. 8, their absorptionand fluorescence spectra as well as their pigment compositionare shown. Despite their different protein-expression profiles,the only significant difference detectable between differentLhcb1 isoforms was a small variation in chlorophyll a/b ratio.Nevertheless, single isoforms do not showproperties that couldjustify a specific functional role thus accounting for differencesin expression level. However, it should be pointed out that inthese experiments we could analyze only isolated monomericcomplexes. Thus, the possibility that the different Lhcb1 couldbe distinct in the framework of supramolecular interactionswithin the photosynthetic membranes could not be verified.

DISCUSSION

Regulation of Antenna Size in Viridis zb63 Mimics the Accli-mation to Extreme High Light—In the mutant viridis zb63 weobserved a strong regulation of the antenna size, as exemplifiedin Fig. 9, which shows the distribution of different pigment-binding complexes in mutant thylakoid membranes as com-pared with WT. It clearly appears that the ratio of antennacomplexes to the PSII core is strongly reduced in the mutant.When the antenna proteins were analyzed in more detail bytwo-dimensional electrophoresis and Western blotting, weshowed that Lhcb1–2-3 and Lhcb6 were the polypeptidesshowing the strongest decrease, whereas Lhcb4 and Lhcb5 con-

tent was essentially stable. Interestingly, this behavior is con-sistent with recent analyses in Arabidopsis thaliana plantsacclimated to high light conditions, where it was observed thatLhcb6 and Lhcb1–2-3 were the major responsible of thechanges in antenna size occurring during acclimation, whereasLhcb4–5 levels weremaintained (3). The regulation of antennasize on viridis zb63, thus, is qualitatively similar but moreextreme with respect to what observed was in Arabidopsisplants treatedwith intense light and low temperatures, suggest-ing regulatory mechanisms involved are very similar. There-fore, results obtained here with viridis zb63 are not due to thespecific mutation but rather reflect the normal regulation ofantenna size in WT plants, making thus its analysis a powerfultool to yield information on antenna size regulation.The use of viridis zb63 as a model system, however, has a

significant advantage, because such a strong effect on theantenna size was achieved without subjecting plants to oxida-tive stress conditions like high light and low temperature. Infact, we verified that the light intensity used in this experimentwas sufficient to reduce the PQ redox pool without generatingoxidative stress, as determined by the quantification of lipidperoxides accumulation (Fig. 1). This analysis of viridis zb63thus allows analyzing the effects of PQ reduction without anyoverlap with those induced by ROS, a distinction that is notpossible in a high light-treated plant where both responses areoverlapping.PQ Redox State Is a Signal for Post-transcriptional Regula-

tion—Photosynthesis is a complex phenomenon, composed bymany reactions of different nature (chemical and physical) thatneed to be synchronized and regulated in response to environ-mental conditions. For instance, the capacity of light absorp-tion must be balanced with the photosystems capacity of usingexcitation energy harvested. The need for such a regulation isdue to the choice of chlorophylls as chromophores for lightharvesting: a porphyrin with long living excited states and hightriplet yield. Because of these properties, energy absorbed inexcess may lead to the formation of harmful ROS. The redoxstate of the electron transport chain between PSI and PSII is anindicator of the overall efficiency of the photosynthesis lightphase, and it was suggested to play a key role in the regulation ofantenna size. In particular, it was shown that PQ redox state isimplicated in the down-regulation of the lhc genes expressionin high light conditions (8).In this work we used the barley mutant viridis zb63, a geno-

typewith a chronically reduced PQ, as amodel for clarifying therole of its redox state in long term regulation of gene expression.By analyzing both gene transcription and polypeptides steady-state levels, we observed that, although polypeptides levels weredrastically affected (14), gene expression showed only smallvariations and restricted to Lhcb1. To understand this apparentdiscrepancywith literature data, it is worth considering that ourwork covered a more extended timescale with respect to previ-ous experiments. In fact, we analyzed plants at the end of 9 daysof chronic plastoquinone reduction, while previous experi-ments suggesting the key role of PQ redox state for lhc geneexpression were performed upon treating with light stress forfew hours (8). A recent study, however, pointed out that, whenlhc expression was analyzed up to 24 h of high light treatment,

FIGURE 7. Western blot titration on viridis 115. Different polypeptides werequantified in WT and viridis 115 thylakoids by detection with specific antibod-ies. For each assay only one dilution is shown in A for clarity, but the quanti-fication reported in B is based on four dilutions for each assay. All valuesreported are normalized to polypeptide content in WT.

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the transcription inhibition was transitory and final mRNA lev-els recovered to roughly the starting levels (13), consistent withour results. This behavior, however, could be alternativelyexplained with a rapid induction of oxidative stress and conse-quently a transient accumulation of ROS, as suggested previ-ously (33): the treatment with norfluorazon, which blocks chlo-roplast development, leads to accumulation of chlorophyll

precursors that easily interact withoxygen, generating ROS, and resultsin the repression of lhcb genes.In our work, instead, we can

exclude ROS effects and confidentlyconclude that lhc gene expressiondoes not play a key role for the reg-ulation of polypeptide accumula-tion during long term acclimation.Furthermore, not only the lhc but allgenes involved in the light phase ofphotosynthesis and encoding thefour large complexes in the thyla-koid membranes (PSI, PSII, cyto-chrome b6f, and ATPase) shared thesame transcriptional behavior. Thissuggests that, although differencesbetween individual genes may existin the short term, long termmecha-nisms induced by the over-reduc-tion of PQ pool do not consist ofstrong transcription inhibition.Despite lack of relevant tran-

scriptional regulation, however,the level of Lhc polypeptides isindeed strongly decreased in themutant with respect to the WT(Figs. 4 and 6). We conclude thatthe steady-state level of Lhc pro-teins depends on post-transcrip-tional rather than on transcriptionalregulation. It is worth emphasizingthat this dependence is reallystrong: in viridis zb63, in fact, theantenna size is reduced tominimumlevel (14) due to strong decrease ofLhcb1–3 and Lhcb6, whereas,according to array analysis, tran-scription is slightly affected in lhcb1only.Several mechanisms could be

responsible for the observed post-transcriptional regulation: first,transcript stability could be de-creased, but such a variation wouldhave been detected in array analysiswhere stationary mRNA levels areanalyzed. Further possible regula-tion mechanisms can instead oper-ate at translation level. In fact, amutant in a RNA-binding protein,

Nab1, was recently shown to affect Lhcb translation inChlamy-domonas (34). In addition, polysome association of photosyn-thetic mRNA, including lhc transcripts, was shown to beaffected by exposure to stress conditions (35). However,according to these data the effect on translation appears to becommon to whole mRNA rather than restricted to photosyn-thesis-related genes.

FIGURE 8. Characterization of Lhcb1 isoforms reconstituted in vitro. One member for each class of Lhcb1isoforms identified from proteomic analyses (lhcb1a– e) was reconstituted in vitro. In the case of class a allputative members were included and indicated as 01 and 02. A, pigment binding properties of refolded com-plexes. Data are normalized to 12 total chlorophylls per molecule as in a previous study (18). B and C, absorptionand fluorescence excitation spectra of refolded complexes. Given that all spectra are very similar, only the onesfor Lhcb1a.01, Lhcb1a.02, Lhcb1b, and Lhcb1d are reported, respectively, in solid, dashed, dotted, and dash-dotted lines.

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An alternative possibility is that protein regulation isachieved by activation of polypeptides degradation. Some find-ings in the literature already suggested thismechanism is activeinmodulating Lhc accumulation: for instance it was shown thattransition from low to high light activates the proteolysis ofLHCII trimer subunits (36, 37). Consistent with this idea, weobserved that the transcription of two different proteases wasinduced in the mutant with respect to WT. One of them playssome role in regulation of photosynthesis, being a chloroplasticClp protease, which was already shown to be active againstOE33 (38).Other chloroplast proteases, belonging to the FtsH family,

were shown to be associated to the thylakoid membrane and tobe active in degrading antenna proteins (39). Their activity ver-sus antenna polypeptides was shown to be influenced by acti-vation of the senescence program (40) or by acclimation to highlight conditions (41). This family of proteases was representedin array data set (see supplementary material), but none of thefive FtsH sequences showed regulation in the mutant withrespect toWT. However, this result was expected, because pre-vious reports already showed that these proteases are not over-expressed in high light (42). On the contrary, activation of pro-teolysis was proposed to be triggered by the exposure of anunfolded region at the N terminus of target proteins (43), as inthe case of photo-oxidative stress (44). We are presently verify-ing the possibility that these proteases are indeed differentlyactivated in viridis zb63 with respect to the WT plants.

A further suggestion that Lhc degradationmight play amajorrole in antenna size regulation in this mutant derives from theobservation that viridis zb63 largely accumulates zeaxanthin inLhc proteins (thylakoids de-epoxidation index is 0.58). In fact, itwas previously shown in the lut2npq2 Arabidopsis mutant,which presents zeaxanthin as the only xanthophyll, that theaccumulation of this carotenoid induces a decrease in antennasize because of selective destabilization of trimeric LHCII (45).Similar results have been reported also in the lor1npq2mutantof C. reinhardtii and thus appears to be a general regulatorymechanism of Lhc proteins (46). Binding of zeaxanthin to Lhcproteins was shown to induce conformational changes, as dem-onstrated by spectroscopic as well as biochemical methods (47,48) and protease-sensitive sites might becomemore exposed asa consequence. Thus, zeaxanthin accumulation in excess lightconditions, in addition to its other effects, could have a photo-protective role by inducing the reduction of antenna size andreducing light-harvesting capacities.Epistatic Regulation of Translation in Higher Plants Is Effec-

tive for Core Complexes but Not for Antenna Proteins—Weobserved little or no effect on the transcription of genes encod-ing the PSI core complex subunits in the viridis zb63 mutant.On the contrary, the corresponding polypeptides were all con-comitantly undetectable. This phenotype could be due to trans-lational regulation, to protein degradation, or both. Previousreports showed the role of assembly factors on protein accumu-lation for both PSI (49) and PSII (50) core complexes in C.reinhardtii and demonstrated the presence of control by epis-tasis of synthesis. It was shown, in fact, that the absence of PsaBcaused a down-regulation of PsaA synthesis due to translationauto-regulation. Similarly, in the case of PSII, the absence of D1or D2 proteins led to inhibition of translation initiation of otherPSII subunits, without alteration of mRNA levels. In differentspecies, however, this regulatory mechanism is not always con-served: in cyanobacteria, in fact, mutations affecting PSIIassembly were shown to increase the proteolytic susceptibilityof other subunits, without alteration of transcription or trans-lation rate (51).In the case of plants direct evidences are still lacking. How-

ever, we consider the hypothesis of an epistatic regulation asbeingmore likely considering themore recent divergence fromgreen algae and the conservation in photosystem organizationin Viridiplantae. Consistently, a recent report in A. thalianasuggested the presence of a translational mechanism similar tothe one evidenced in algae: the mutant lpa1 accumulates lowerlevel of PSII proteins due to a strong reduction in translation,despite normal RNA level and polysome association (52). Inaddition, very recently, control by epistasis of synthesis hasbeen shown to play amajor role in tobacco at least in the case ofRubisco large subunit (53). The composition of photosyntheticapparatus observed in viridis zb63mutant is consistentwith thehypothesis that a similar mechanism of epistatic regulation isactive in PSI and PSII assembly in higher plants, although pro-tein degradation cannot be ruled out.In addition to previous works, moreover, this study also

allowed insights into the assembly of antenna proteins in pho-tosystems. In fact, while we observed such a regulation (eitherdue to epistasis or protein degradation) in the case of core pro-

FIGURE 9. Non-denaturing Deriphat-PAGE of WT and viridis zb63 (zb63)thylakoid membranes. 30 �g of chlorophylls was loaded for each sample.

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teins, specific Lhc subunits were maintained in both mutantsaffected in PSI or PSII core complexes, although the overallpool of antenna proteins was reduced. These antenna proteinswere not only present but also properly folded, as shown in thecase of viridis zb63, where the peculiar red fluorescence typicalof PSI antenna is detected despite the absence of PSI core (14).The evidence for a distinctmode of regulation for core versus

antenna assembly is even more striking if we consider the caseof Lhcb6, a PSII subunit that is reduced in the PSII-less mutantviridis 115 while completely absent in viridis zb63. This sug-gests the presence of a finely regulated process: Lhcb6 is theantenna protein undergoing the strongest changes in abun-dance in response to growth conditions (3); its reduction inhigh light might trigger disconnection and degradation ofperipheral LHCII population leading to reduction in antennasize. This down-regulation is extreme in viridis zb63, becauseLhcb6 polypeptide is undetectable; on the contrary, in viridis115, despite the absence of a functional PSII, the light excessexperienced by the cells is not high enough to drive its completedeletion.In conclusion, our analysis suggests that antenna proteins are

independently folded in the thylakoid membranes and onlysuccessively associated to the assembled core complex. If thisassociation fails, antenna proteins undergo proteolytic degra-dation, but their synthesis and assembly are not prevented. Thisdistinct regulation of core and antenna subunit assemblyappears to be a commonmechanism of all antenna proteins forboth PSI and PSII, because viridis zb63 and viridis 115 showeda common phenotype in this respect.Role of Different Specific Lhcb Polypeptides—In viridis zb63

we observed a strong regulation of Lhcb1–2-3 and Lhcb6 con-tent, whereas Lhcb4–5 are essentially maintained. This obser-vation is consistent with a previous report (14) showing that theminimal PSII antenna is composed by a LHCII trimer (S trim-ers, from Strongly bound (54)), Lhcb5 and Lhcb4, while Lhcb6is absent. Here we also present evidences that Lhcb3 as well isabsent in the LHCII trimers (S trimers) closely connected toPSII core and maintained in viridis zb63 (54), whereas it isincluded inMtrimers, where it participates to anLhcb4-Lhcb6-LHCII complex (55, 56).It is also worth observing that in viridis zb63 some Lhcb1

isoforms have a specific regulatory behavior, despite their verysimilar polypeptide sequences. This is consistent with similarregulation of different Lhcb1 polypeptides in maize acclimatedto different light conditions (57). We suggest that these con-served Lhcb1 isoforms are preferential components of theLHCII trimers in S position that are retained in themutant (14).Despite this conserved regulation, however, the monomericpigment-protein complexes refolded in vitro did not show dif-ferences in their biochemical and spectroscopic properties.This is consistent with their high sequence homology, and itsuggests that the intrinsic function in light harvesting and pho-toprotection is the same. It cannot be excluded, however, thatthe very small differences in primary sequence may cause dif-ferential accessibility for LHCII-degrading proteases, alsobecause of variable interaction with other subunits, like Lhcb5or Lhcb4. One further possibility to explain the reason for thedifferent regulation of Lhcb1 isoforms can be inferred from the

analysis of Lhcb sequences in different organisms: sequenceanalyses show that the presence of multiple isoforms of LHCIIcomponents is a conserved feature in the green lineage (58).However, these isoforms show conserved species-specific char-acteristics in sequence properties rather than conserved fea-tures between different species. This suggests that there mightbe a selective advantage in having multiple copies of very simi-lar genes. One possible explanation may be proposed on thebasis of the dual role of Lhc proteins in light harvesting andphotoprotection. Lhcb1, as mentioned are down-regulated inhigh light condition, leading to a reduction of the antenna size.The presence of few, similarly regulated, Lhcb subunits wouldlead to complete degradation of the antenna system understrong high light, thus leaving PSII core complex with littleprotection as in the ch1mutant (59). On the contrary, multipleLhcbs with distinct sensitivity to degradation still provide thepossibility ofmodulating antenna size while ensuring themain-tenance of photoprotection: in viridis zb63, in fact, a minimalantenna size is retained even in extreme stress conditions (14).

Acknowledgment—We thank Dr. Stefano Caffarri (LGBP, Universitede Marseille) for Lhcb1 cloning.

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BassiFriedel Drepper, Wolfgang Haehnel, Luigi Cattivelli, Tomas Morosinotto and Roberto Sara Frigerio, Chiara Campoli, Simone Zorzan, Luca Isaia Fantoni, Cristina Crosatti,

Redox State at the Post-transcriptional Rather than Transcriptional LevelPhotosynthetic Antenna Size in Higher Plants Is Controlled by the Plastoquinone

doi: 10.1074/jbc.M705132200 originally published online August 5, 20072007, 282:29457-29469.J. Biol. Chem. 

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